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Genomic restructuring was detected in newly synthesized tritordeum by molecular and cytogenetic tools. Genomic stability is expected for advanced tritordeum lines (HHAABB; 2n = 42) with multiple generations of self-fertilization. This study intends to confirm or decline this hypothesis by characterizing three advanced tritordeum lines and their parental species using cytogenetics, inter-simple sequence repeat (ISSR) and retrotransposon-based markers. Mitotic chromosomes of each tritordeum line were hybridized with six synthetic oligonucleotide probes using non-denaturing fluorescence in situ hybridization. Polymorphic hybridization patterns and structural rearrangements involving SSR regions were detected. The same chromosome spreads were re-hybridized with genomic DNA of Hordeum chilense Roem. et Schult. and the 45S ribosomal DNA (rDNA) sequence pTa71. These FISH experiments allowed for parental genome discrimination, identification of nucleolar chromosomes, and detection of structural rearrangements, mostly involving rDNA loci. The chromosomes bearing SSR hybridization signals and/or chromosomes involved in structural rearrangements were identified. ISSR, retrotransposon-microsatellite amplified polymorphism, inter-retrotransposon amplified polymorphism and inter-primer binding site markers evidenced genomic reshuffling in all tritordeum lines relative to their parents. Line HT28 was considered the most genetically stable. This work demonstrated that cytogenetic and molecular monitoring of tritordeum is needed, even after several self-fertilization generations, to guarantee the selection of the most stable lines for improvement and sustainable agriculture. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction-amplified product length polymorphism (PCR-APLP) is one of the most convenient and reliable methods for single nucleotide polymorphism (SNP) analysis. This method is based on PCR, but uses allele-specific primers containing SNP sites at the 3′-terminus of each primer. To use this method at least two allele-specific primers and one “counter-primer”, which serves as a common forward or reverse primer of the allele-specific primers, are required. The allele-specific primers have SNP sites at the 3′-terminus, and another primer should have a few non-complementary flaps at the 5′-terminus to detect SNPs by determining the difference of amplicon length by PCR and subsequent electrophoresis. A major disadvantage of the addition of a non-complementary flap is the non-specific annealing of the primer with non-complementary flaps. However, a design principle for avoiding this undesired annealing has not been fully established, therefore, it is often difficult to design effective APLP primers. Here, we report allele-specific primers with an inosine chain at the 5′-terminus for PCR-APLP analysis. This unique design improves the competitiveness of allele-specific primers and the reliability of SNP analysis when using the PCR-APLP method. [ABSTRACT FROM AUTHOR]

A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements. [ABSTRACT FROM AUTHOR]